1. Volume 141, Issue 1, Pages 176-185 (July 2011)
MyD88 and Retinoic Acid Signaling Pathways Interact to Modulate Gastrointestinal Activities of Dendritic Cells 
Eduardo J. Villablanca, Sen Wang, Jaime de Calisto, Daniel C.O. Gomes, Maureen A. Kane, Joseph L. Napoli, William S. Blaner, Hiroyuki Kagechika, Rune Blomhoff, Mario Rosemblatt, Maria Rosa Bono, Ulrich H. von Andrian, J. Rodrigo Mora 
Gastroenterology 
Volume 141, Issue 1, Pages (July 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
DC ability to imprint gut homing correlates with retinoid levels in the gut. (A) Quantification of retinyl esters, retinol, and RA in duodenum (duo), jejunum (jej), ileum (ile), and colon (n = 6). (B) LP-DC or PP-DC from duo, jej, ile, and colon were used to activate naïve CD8 T cells. After 4−5 days, T cells were analyzed for α4β7 and CCR9 expression (n = 5). (C) DC were used to activate naïve CD4 T cells. After 4 days, T cells were analyzed for Foxp3 expression. Fluorescence-activated cell sorting (FACS) plots are representative of 2 independent experiments. (D) Retinal dehydrogenase (RALDH) activity in DC (n = 4). (E) Luciferase activity in DC from DR5-luciferase mice (n = 4). Mean ± SEM; *P < .05; **P < .01; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
RA is necessary in vivo for gut-associated DC education. DC were isolated from mice on a VAD or control diet. (A) DC were used to activate naïve CD8 T cells. After 4−5 days, T cells were analyzed for α4β7 and CCR9 expression (n = 4). (B) DC were used to activate naïve CD4 T cells. After 4−5 days, T cells were analyzed for Foxp3 expression. FACS plots are representative of 2 independent experiments. (C) DC were cocultured with naïve B cells activated with anti-IgM plus IL-5. After 4 days, cocultures were analyzed for intracellular IgA in B220Int cells and IgA in the supernatant (n = 4). (D) Aldh1a2 mRNA and (E) RALDH activity in CD11c+ and CD11c+CD103+ DC (n = 5). (F) RALDH activity in MLN CD11c+ cells from control or VAD mice ± oral RA supplementation. FACS plots are representative of 2 independent experiments. Mean ± SEM; *P < .05; **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
RA is sufficient to confer DC with RA synthesizing capacity in vitro. (A) Aldh1a2 mRNA in spleen-DC incubated for 24 hours with 100 nM all-trans RA or 100 nM of the indicated nuclear receptor agonists. HX630 and PA024 were used at 1 or 10 μM, with similar results (n = 5−10). (B−F) spleen-DC were incubated for 24 hours with or without 100 nM RA (RA-DC and UT-DC, respectively) and analyzed for (B) RALDH activity (n = 5) or (C) washed, pulsed with antigen, and used to activate naïve CD8 T cells from T-cell receptor transgenic OT-1xRAG2−/− mice in fetal bovine serum−free media ± 50 nM retinol. After 4−5 days, T cells were analyzed for α4β7 and CCR9 expression (n = 7). (D) Cocultures were performed as described in (C) either in the presence or absence of the RALDH inhibitor diethylaminobenzaldehyde (DEAB) (n = 7). (E) Competitive homing experiment between CD8 T cells activated with RA-DC or UT-DC (n = 5). (F) Naïve CD4 T cells from OT-2xRAG2−/− mice were activated with UT-DC or RA-DC. Five days later CD4 T cells were analyzed for Foxp3 expression (n = 3).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
RA is sufficient to confer DC with RA synthesizing capacity in vivo. (A) Wild-type mice were supplemented orally with RA (400 μg/dose) every day for 5 days. After that, MLN-DC and PLN-DC were isolated and analyzed for the expression of (B) Aldh1a2 mRNA and (C) RALDH activity (n = 5). (D) PLN-DC from control or RA-treated mice were used to activate naïve CD8 T cells in fetal bovine serum−free media ± retinol and either in the presence or absence of DEAB. FACS plots are representative of 2 independent experiments. Mean ± SEM; *P < .05; **P < .01; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
DC education requires ERK/MAPK signaling. (A−C) Spleen-DC were incubated for 24 hours with or without 100 nM RA and in the presence or the absence of inhibitors for p38 (SB203580, 10 μM), ERK (U0126, 10 μM), nuclear factor−κB (SN50, 50 μM), or c-Jun-N-terminal kinase (SP600125, 50 μM). (A) DC were used to activate naïve CD8 T cells. After 4−5 days, T cells were analyzed for α4β7 and CCR9 expression (n = 9). (B) Aldh1a2 mRNA expression in DC (n = 3). (C) Spleen-DC from DR5-luciferase mice were incubated for 24 hours with or without RA and in the presence or absence of the ERK inhibitor (U0126, 10 μM) and analyzed for their luciferase activity (n = 3). (D) Wild-type mice were orally treated with the ERK inhibitor PD (25 μg/g/dose) every other day for 6 days. After that, CD11c+ MLN-DC were analyzed for CD103 expression and RALDH activity (n = 5). Mean ± SEM; *P < .05; **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Figure 6
MyD88 is required for RA-mediated DC education. (A, B) Wild-type or MyD88−/− spleen-DC were incubated with 100 nM RA for 24 hours. (A) DC were used to activate naïve CD8 T cells. After 4−5 days, T cells were analyzed for α4β7 and CCR9 expression (n = 5). (B) Aldh1a2 (n = 4) or Tgm2 (n = 5) mRNA expression in DC. (C) Wild-type or MyD88−/− mice were supplemented orally with RA (400 μg/dose) every day for 5 days and PLN-DC were analyzed for RALDH activity (n = 7). (D) Spleen-DC from wild-type or MyD88−/− mice were analyzed for their expression of Rxra, Rxrb. Rxrg, Rara, Rarb, and Rarg mRNA (n = 4−6). ND, not detected. (E) Spleen-DC were incubated for 24 hours with or without 100 nM RA and either in the presence or absence of the RARβ inhibitor LE540 (1 μM) and then analyzed for Aldh1a2 mRNA expression (n = 3). (F) Spleen-DC from DR5-luciferase mice were incubated for 24 hours with or without 100 nM RA and either in the presence or absence of LE540 and then analyzed for their luciferase activity (n = 3). Mean ± SEM; *P < .05; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

8. Figure 7
TLR signals contribute to RA-mediated human and murine DC education. (A) Mo-DC from human healthy donors were treated with either RA (100 nM), Pam3CSK4 (0.5 μg/mL), or both from day 3 of differentiation. RALDH activity was analyzed at day 6 in CD11c+ cells (n = 4). FACS plots show representative results in Mo-DC from 2 donors. (B) Mo-DC pretreated with either RA, Pam3CSK4, or both, were washed and cultured with allogenic CD4 T cells activated with anti-CD3 plus anti-CD28 and transforming growth factor−β1 (TGF-β1; 2 ng/mL). After 4 days, CD4 T cells were analyzed for Foxp3 expression. FACS plots show 1 representative experiment out of 2. (C) Spleen-DC were incubated for 24 hours in the presence of 100 nM RA, Pam3CSK4 (0.5 μg/mL), or both, washed and used to activate naive CD8 T cells. After 4 days, T cells were analyzed for their expression of α4β7 and CCR9. FACS plots are representative of 3 experiments. (D) Human Mo-DC were treated at day 6 with or without 100 nM of RA, Pam3CSK4 (0.5 μg/mL), or both, for 24 hours, washed and cocultured with total human T cells activated with plate-bound anti-CD3 plus anti-CD28 antibodies. After 6 days, CD4 and CD8 T cells were analyzed for their expression of α4β7 (n = 3). (E) Spleen-DC were incubated for 24 hours in the presence of 100 nM RA, Pam3CSK4 (0.5 μg/mL), or both, washed and cocultured with naïve B cells activated with anti-IgM plus IL-5. After 4 days, IgA levels were measured in the culture supernatants (n = 7). Mean ± SEM; *P < .05; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions